Aqueous sustained-release formulations of proteins

ABSTRACT

The present invention is directed to sustained-release pharmaceutical formulations of therapeutic proteins containing carboxymethyl ether cellulose polymer and methods of manufacture and use thereof.

FIELD OF THE INVENTION

[0001] The present invention provides aqueous sustained-releasepharmaceutical formulations of therapeutic proteins containingcarboxymethyl ether cellulose polymer and their method of manufacture.The present invention also provides methods to use the pharmaceuticalformulations that provide a variety of new benefits, including greaterefficacy, safety, patient convenience, and patient compliance.

BACKGROUND OF THE INVENTION

[0002] Due to recent advances in genetic and cell engineeringtechnologies, proteins known to exhibit various pharmacological actionsin vivo are capable of production in large amounts for pharmaceuticalapplications. A major limitation of the development of proteintherapeutics is the preparation of stable pharmaceutical formulations ofthe proteins. Therapeutic proteins are typically administered byfrequent injection because the active agent protein generally has shortin vivo half-lives and negligible oral bio-availability, thus posing asignificant physical burden on the patient and associated administrativecosts. As such, there is currently a great deal of interest indeveloping and evaluating sustained-release formulations. Effectivesustained-release formulations can provide a means of controlling bloodlevels of the active ingredient, and also provide greater efficacy,safety, patient convenience and patient compliance.

[0003] To date there have been mainly two mechanisms to achievesustained-release of a protein therapeutic: 1) modifying the protein toincrease the half-life of the protein, typically by reducing theclearance rate; and 2) preparing sustained-release formulations ofprotein encapsulated in polymer microspheres. The advantage togenerating sustained release formulations is that the formulation couldtheoretically be utilized for many protein therapeutics.

[0004] Examples of polymer microsphere sustained release formulationsare described in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat.Nos. 5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).

[0005] U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO96/40073 describe a polymeric matrix containing particles oferythropoietin that are stabilized against aggregation with a salt.

[0006] Unfortunately, the instability of most proteins (e.g.denaturation and loss of bioactivity upon exposure to heat, organicsolvents, etc.) has greatly limited the development and evaluation ofsustained-release formulations thereof. In addition these techniques arenot as broadly applicable as generally anticipated due to thebiochemical variability in protein that greatly influences anyindividual protein tolerance for the conditions required to producemicrospheres.

[0007] PCT publication WO 00/38651 describes a pharmaceuticalcomposition containing a protein in a polymeric matrix that hasthermally and pH responsive gelation/de-gelation properties. Theseformulations may be prepared without exposure to heat or organicsolvents, but are characterized by their use of modified hydrogels. Theuse of thermostable hydrogels suffers by undesirable difficulties ofmanufacture and thus is currently commercially impractical. Anotherundesirable characteristic of these modified hydrogels is that theirbiocompatibility or immunogenicity is poorly characterized.

[0008] U.S. Pat. No. 4,717,717 to A. L. Finkenaur describes compositionsof epidermal growth factor that are stabilized against loss ofbiological activity by the presence of a cellulose polymer. There is nodescription of in vivo sustained release properties of theseformulations.

[0009] U.S. Pat. No. 5,457,093 to Cini et al. describes gel formulationscontaining growth factors used in ophthalmic and topical applications.There is no description of the use of these formulations for parenteraladministration, nor is there any suggestion that these formulationswould have sustained release properties in vivo.

[0010] There remains a need, therefore, for parenteral sustained-releaseformulations of erythropoietin that can be manufactured easily andpromote the stability of active agent protein contained therein.

SUMMARY OF THE INVENTION

[0011] The present invention provides a method to prepare aqueoussustained-release pharmaceutical formulations of therapeutic proteinsfor parenteral administration. The present invention also providespharmaceutical formulations for general use with proteins for parenteraladministration comprising:

[0012] a) a pharmaceutically active amount of a protein;

[0013] b) a pharmaceutically acceptable pH buffering agent to provide apH in the range of about pH 4.5 to about pH 9;

[0014] c) a tonicity agent in the concentration range of about 0 toabout 125 millimolar; and

[0015] d) Sodium carboxymethyl ether cellulose in the concentrationrange of about 0.5% to about 7% total formula weight;

[0016] wherein the pH of the aqueous formula is about 4.5 to about pH9.0.

[0017] A preferred formulation of the present invention provides apharmaceutical formulation of erythropoietin comprising:

[0018] a) a pharmaceutically active amount of erythropoietin;

[0019] b) a pharmaceutically acceptable pH buffering agent to provide apH in the range of about pH 6 to about pH 9;

[0020] c) a tonicity agent in the concentration range of about 0 toabout 200 millimolar; and

[0021] d) sodium carboxymethyl ether cellulose (CMC) in theconcentration range of about 0.5% to about 7% total formula weight, saidCMC having a molecular weight in the range of about 50,000 daltons toabout 1,000,000 daltons;

[0022] wherein the pH of the formulation is about pH 6.3 to about pH8.3.

[0023] The present invention also provides a method to prepare aqueoussustained-release pharmaceutical formulations of erythropoietincomprising admixing an aqueous solution of a CMC polymer with apharmaceutically active amount of EPO. The formulations of the presentinvention utilize CMC polymer that is primarily in a solution staterather than in a micro-particulate state.

[0024] The present invention also provides methods to use thesesustained-release formulations comprising dosing regimens where theformulation is administered at a wide variety of desired intervals,including, but not limited to, thrice per two weeks, once per week, onceper two weeks, once per three weeks, once monthly, once per five weeks,once per six weeks, or at any other time interval or combination of timeintervals that may be desirable for the particular patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1: Pharmacokinetics of sustained-release formulations CMC-1,CMC-2, CMC-3, CMC-4 illustrated as mean Plasma Epogen Alfa concentrationversus time.

[0026]FIG. 2: Pharmacodynamics of sustained-release formulations CMC-1,CMC-2, CMC-3, CMC-4 illustrated as change in the percentagereticulocytes versus time.

[0027]FIG. 3: Pharmacodynamics of sustained-release formulations CMC-1,CMC-2, CMC-3, CMC-4 illustrated as change in the amount of Hemoglobinversus time.

[0028]FIG. 4: Pharmacodynamics of sustained-release formulations CMC-1,CMC-2, CMC-3, CMC-4 illustrated as change in the number of red bloodcells versus time.

[0029]FIG. 5: Pharmacokinetics of Clinical Batch CMC-EPO illustrated asmean Plasma Epoetin Alfa concentration versus time.

[0030]FIG. 6: Pharmacodynamics of Clinical Batch CMC-EPO illustrated aschange in the percentage of reticulocytes versus time.

[0031]FIG. 7: Pharmacodynamics of Clinical Batch CMC-EPO illustrated aschange in the amount of Hemoglobin versus time.

[0032]FIG. 8: Pharmacodynamics of Clinical Batch CMC-EPO illustrated aschange in the number of red blood cells versus time.

DETAILED DESCRIPTION OF THE INVENTION

[0033] As used herein, “erythropoietin” or “EPO” shall include thosepolypeptides and proteins that have the biological activity ofrecombinant human erythropoietin (rhEPO), as well as erythropoietinanalogs, erythropoietin isoforms, erythropoietin mimetics,erythropoietin fragments, hybrid erythropoietin proteins, fusionproteins oligomers and multimers of the above, homologues of the above,glycosylation pattern variants of the above, and muteins of the above,regardless of the biological activity of same, and further regardless ofthe method of synthesis or manufacture thereof including, but notlimited to, recombinant (whether produced from cDNA or genomic DNA),synthetic, transgenic, and gene activated methods. Specific examples oferythropoietin include, Epoetin alfa (EPREX®, ERYPO®, PROCRIT®), novelerythropoiesis stimulating protein (NESP™, ARANESP™, darbepoetin alfa)such as the hyperglycosylated analog of recombinant human erythropoietin(Epoetin) described in European patent application EP640619, humanerythropoietin analog (such as the human serum albumin fusion proteinsdescribed in the international patent application WO9966054),erythropoietin mutants described in the international patent applicationWO9938890, erythropoietin omega, which may be produced from an Apa Irestriction fragment of the human erythropoietin gene described in U.S.Pat. No. 5,688,679, altered glycosylated human erythropoietin describedin the international patent application WO9911781 and EP1064951, PEGconjugated erythropoietin analogs described in WO9805363 or U.S. Pat.No. 5,643,575. Specific examples of cell lines modified for expressionof endogenous human erythropoietin are described in international patentapplications WO9905268 and WO9412650. The generally preferred form ofEPO is purified recombinant human EPO (rhEPO), currently formulated anddistributed under the trademarks of EPREX®, ERYPO®, PROCRIT® orARANESP™.

[0034] As used herein, the term “protein” includes peptides,polypeptides, consensus molecules, analogs, derivatives or combinationsthereof. The term “protein” embraces polypeptide sequences containingmodified amino acids and glycoproteins, or proteins that contain atleast one serine, threonine, or arginine side chain bearing acarbohydrate moiety. Also included are those polypeptides with aminoacid substitutions that are “conservative” according to acidity, charge,hydrophobicity, polarity, size or any other characteristic known tothose skilled in the art. See generally, Creighton, Proteins, W. H.Freeman and Company, NY (1984) pp. 498. Generally one may make one ormore changes in selected amino acids so long as such a change preservesthe overall biological activity of the protein. Small amino terminalextensions, such as an amino-terminal methionine or serine residue, asmall linker peptide of up to about twenty to twenty-five residues, or asmall extension that facilitates purification, such as a poly-histidinetract, an antigenic epitope or a binding domain, may also be present.See, in general, Ford et al., Protein Expression and Purification (1991)2:95-107. Polypeptides or analogs thereof may also contain one or moreamino acid analogs, such as peptidomimetics. One skilled in the art willreadily be able to adapt a desired protein active agent to thecompositions of present invention.

[0035] The term “subject” as used herein, refers to an animal,preferably a mammal, most preferably a human, who is the object oftreatment, observation or experiment.

[0036] The amount of protein used in the formulations of the presentinvention will vary with the biological potency of the protein as wellas the desired potency of the formulation, but will generally containabout 1 μg/ml to about 2000 μg/ml protein per formulation. Specificallythe erythropoietin-containing formulations of the present invention maycontain a “pharmaceutically active amount of erythropoietin”, generallyabout 1000 IU/ml to about 180,000 IU/ml of erythropoietin, wherein120,000 IU is approximately 1000 μg. The erythropoietin may be providedas an aqueous solution of a bulk reagent that is diluted into theformulation of the present invention or may be provided as a driedreagent and reconstituted using the appropriate amount of the aqueousformulation. Dried reagents include, for example, lyophilized orspray-dried erythropoietin. Where erythropoietin is provided as a bulkreagent in formulations of high potency (e.g. greater than 100,000IU/ml), it is preferable that the erythropoietin bulk reagent beprovided in a phosphate buffered solution. This is due to increasedpatient discomfort caused by high concentrations of citric acid bufferstypically used in the preparation of recombinant human erythropoietin.Buffer exchange is achieved using methods well known in the art, such asdiafiltration or dialysis to provide an EPO bulk that contains less than1 millimolar citrate.

[0037] The amount of buffering agent useful in the pharmaceuticalcompositions of the present invention depends largely on the particularbuffer used and the desired pH of the formulation. The concentration ofbuffering ions will generally range from about 10 mM to about 30 mM.Suitable buffer systems to maintain the pH range of about four to aboutnine include, but are not limited to, sodium citrate/citric acid, sodiumacetate/acetic acid, sodium or potassium phosphate dibasic/monobasic,and any other pharmaceutically acceptable pH buffering agent(s) known inthe art. The use of a buffer system of sodium phosphate dibasic andsodium phosphate monobasic is preferred. A pH-adjusting agent such as,but not limited to, hydrochloric acid, citric acid, sodium hydroxide, ora salt of any of these, in particular sodium citrate, may be added tothe formulations to adjust the formulation pH to within the desiredformulation pH range. One goal for these formulations is to minimize thepatient discomfort associated with subcutaneous administration of thecitrate-buffered formulations. Therefore phosphate buffer systems areparticularly preferred in all formulations of the present invention,both in the aqueous protein bulk reagent and in the formulation buffercomponent.

[0038] The preferred pH range for the protein-containing formulations ofthe present invention is between about pH 4.5 to about pH 9, preferablyin the range of about pH 6 to about pH 7.5. The preferred pH range forthe erythropoietin-containing formulations of the present invention isbetween about pH 6.5 to about pH 8, preferably in the range of about pH6.9 to about pH 7.4.

[0039] One or more ionic tonicity agents may be used in the formulationsof the present invention. An ionic tonicity agent is any agent capableof rendering the formulations of the present invention iso-osmotic ornearly iso-osmotic with human blood and carries a positive or negativecharge in aqueous solutions. Typical suitable tonicity agents are wellknown in the art, and include but are not limited to sodium chloride,potassium chloride, ammonium sulfate, glycine, or other amino acids. Thepreferred tonicity agents of the present invention include, but are notlimited to, NaCl, KCl, and glycine, said agent being used at aconcentration in the range of about 0 to about 170 millimolar. Use ofsodium chloride as a tonicity agent is preferred in the formulations ofthe present invention at a concentration of about 75 mM to about 100 mM.The type of tonicity agent and its concentration may influence thesustained-release properties of the formulation. In formulationscontaining more than one tonicity agent, the total concentration oftonicity agents is generally less than 200 mM.

[0040] Sodium carboxymethyl ether cellulose (CMC-CAS # 9004-32-4) havinga molecular weight of about 50,000 to 1,000,000 is used in theformulations of the present invention in the concentration range of 0.5%to about 7% total formula weight, preferably from about 0.5% to about2%, and most preferably at about 2%. The concentration of CMC used isvaried based on the identity of and amount of the protein used in theformulation. The sustained-release activity of the formulation is testedby comparing pharmacokinetic properties of a CMC containing formulationto an otherwise identical formulation lacking CMC. Formulationscontaining 0.5% to about 2% CMC are generally preferred due to theirease of manufacture and because of the ease of administration byinjection. Generally there is a relationship between the amount ofprotein in the formulation, the amount of CMC in the formulation, andthe pharmacokinetic properties of the formulation. Formulationscontaining more protein require higher amounts of CMC to providesustained release properties. The amount of protein/CMC may be expressedas a ratio of protein (in micrograms) to % CMC (grams per 100 mL).Preferred formulations of EPO contain a ratio less than or equal to 660μg EPO/% CMC.

[0041] The phrase “sustained release” as used herein refers tobeneficial pharmacokinetic properties of the formulation.Pharmacokinetic parameters may be calculated using methods known in theart or as described herein. For example, but not by way of limitation,one or more pharmacokinetic parameters may be calculated by modelindependent methods using WinNonlin software, Version 1.1 (ScientificConsulting, Incorporation, Apex, N.C.). Various pharmacokineticproperties may be considered when evaluating the sustained-releaseproperties of a formulation of the present invention. For example, butnot by way of limitation, the following PK parameters may be evaluatedto determine the sustained-release properties of the formulations of thepresent invention:

[0042] Peak serum concentration (C_(max)): The observed maximum serumconcentration of the protein. A sustained-release formulation may have alower C_(max) than a similar non-sustained release formulation due toslower absorption into circulation.

[0043] Time to C_(max) (t_(max)): The time at which C_(max) occurs. Asustained-release formulation may have a longer T_(max) than a similarnon-sustained release formulation due to slower diffusion intocirculation.

[0044] Terminal half-life (t_(1/2)): A sustained release formulation mayhave a longer t_(1/2) than a similar non-sustained release formulation.

[0045] Without being limited by theory, the inventors contemplate thatthe sustained-release properties of these formulations occur through acombination of hydrophobic interaction of the protein with the CMC andan ionic interaction between the protein and CMC. At the pH described,both the protein and the CMC carry a negative charge, and thus anothercomponent, possibly the charged tonicity agent, forms an “ionic bridge”,similar to a salt-bridge as is well-known in biochemical interactions.The interaction between the protein, CMC and/or the ionic tonicity agentis sufficiently strong to retard diffusion of the protein from theinjection site without being permanently retained at the site. It isreadily apparent to those of ordinary skill in the art that theformulations of the present invention are generally applicable toparenteral administration of therapeutic proteins other thanerythropoietin, including, but not limited to, interferons, granulocytecolony stimulating factor, insulin, antibodies and antibody fragments,somatotropin, tissue plasminogen activator, interleukins, and antigensfor immune responses. The formulations of the present invention utilizeCMC polymer that is primarily in a solution state rather than in amicro-particulate state, however a wide variety of ratios ofmicro-particulate CMC polymer to non-microparticulate CMC polymer may besuitable for use in the formulations of the present invention.

[0046] Proteins and polypeptides suitable for use in the presentinvention include, but are not limited to, insulin, motilin, gastrin,prolactin, adrenocorticotropic hormone (ACTH), erythropoietin, growthhormone (GH), kerantinocyte growth factor (KGF), stem cell factor (SCF),thrombopoietin, osteoprotegerin (OPG), and obesity protein (OB protein:protein may also be referred to herein as leptin), granulocytecolony-stimulating factor (G-CSF), alpha interferon (in particular alpha2 b), beta interferon (in particular beta 1 a and beta 1 b), gammainterferon, interleukin 2, fibroblast growth factors (FGF), insulin-likegrowth factors (IGFs), macrophage colony stimulating factor (M-CSF),granulocyte macrophage colony stimulating factor (GM-CSF), colonysimulating growth factors (CSFs), tumor necrosis factor (TNF), thyroidstimulating hormone (TSH), luteinizing hormone (LH), folliclestimulating hormone (FSH), human chorionic gonadotropin (HCG),neurotrophic growth factor (NGF), neurotrophic factor 3 (NT3),neurotrophic factor 4 (NT4), brain-derived neurotrophic factor (BDNF),glial cell line derived neurotrophic factor (GDNF), platelet-derivedgrowth factor (PGDF: also known as interleukin 11), vascular endothelialgrowth factor (VEGF), bone morphogenetic protein (BMP), megakaryocytegrowth differentiation factor (MGDF), Factor VII, Factor VIIa, FactorVIII, Factor IX, superoxide dismutase (SOD), tissue plasminogenactivator (TPA), urokinase, streptokinase, kallikrein,alpha-galactosidase, Pancreatic Rnase, platelet activting factoracetylhydrolase, interleukin-1 receptor antagonist (IL-Ira), REMICADE(Infliximab: a monoclonal antibody that blocks the biological activityof circulating TNFα ENBREL (etanercept: dimeric fusion proteinconsisting of the extracellular ligand-binding portion of the human 75kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to Fcportion of human IgGl).

[0047] The formulations of the present invention are prepared byadmixing the formulation reagents in an aqueous solution such that thecomponents are mixed substantially uniformly so that none of thecomponents are localized. Advantageously all of the formulationcomponents, except the protein component, can be prepared and adjustedto conditions suitable for the protein prior to the addition of theprotein component. Alternatively, the protein bulk reagent may bediafiltered into an appropriate buffer system, preferably phosphatebuffer, and the other reagents may be added to the protein bulk, and thebulk protein concentration can be adjusted appropriately to the desiredpotency.

[0048] A preferred method of formulation for erythropoietin-containingformulations of the present invention, as well as otherprotein-containing formulations generally, comprises the steps:

[0049] a) difiltering a recombinant human EPO bulk solution against a 10to 30 millimolar phosphate buffer to provide a phosphate buffered EPObulk containing less than 1 mM citric acid;

[0050] b) admixing a quantity of CMC to the phosphate buffered EPO bulksufficient to provide a final concentration of about 0.5% to about 7%CMC;

[0051] c) admixing a quantity of NaCl to the phosphate buffered EPO bulksufficient to provide a final concentration of about 0 mM to about 170mM NaCl; and

[0052] d) adjusting the EPO bulk with water sufficient to provide apredetermined final formulation volume and EPO potency.

[0053] The formulations of the present invention are administered to asubject in need thereof via parental administration excludingintravenous administration. Particular routes of parenteraladministration include, but are not limited to, intramuscular,subcutaneous, intraperitoneal, intracerebral, intraventricular,intracerebroventricular, intrathecal, intracisternal, intraspinal and/orperi-spinal routes of administration by delivery via intracranial orintravertebral needles and/or catheters with or without pump devices.The route of administration may be selected based on the therapeuticindication of the pharmaceutically active protein.

[0054] Hormones, cytokines, coagulation factors, and other biologicallyactive proteins including insulin, motilin, gastrin, prolactin,adrenocorticotropic hormone (ACTH), erythropoietin (EPO), growth hormone(GH), stem cell factor (SCF), thrombopoietin, osteoprotegerin (OPG), andobesity protein (OB protein: OB protein may also be referred to hereinas leptin), granulocyte colony-stimulating factor (G-CSF), alphainterferon (in particular alpha 2 b), beta interferon (in particularbeta 1 a and beta 1 b), gamma interferon, interleukin 2, insulin-likegrowth factors (IGFs), macrophage colony stimulating factor (M-CSF),granulocyte macrophage colony stimulating factor (GM-CSF), colonysimulating growth factors (CSFs), tumor necrosis factor (TNF), thyroidstimulating hormone (TSH), luteinizing hormone (LH), folliclestimulating hormone (FSH), human chorionic gonadotropin (HCG) vascularendothelial growth factor (VEGF), megakaryocyte growth differentiationfactor (MGDF), Factor VII, Factor VIIa, Factor VIII, Factor IX,superoxide dismutase (SOD), tissue plasminogen activator (TPA),urokinase, streptokinase, kallikrein, alpha-galactosidase, PancreaticRnase, platelet activting factor acetylhydrolase, interleukin-1 receptorantagonist (IL-Ira), REMICADE and ENBREL may be administered viaintramuscular, subcutaneous, or intraperitoneal routes such that thereis a systemic release of the protein.

[0055] Certain growth factors, such as kerantinocyte growth factor(KGF), fibroblast growth factors (FGF), platelet-derived growth factor(PGDF: also known as interleukin 11), may be administered at or near asite of action to achieve a localize benefit. Local injection of theformulation of the present invention may result in higher local-areaconcentrations of the active pharmaceutical reagent and may be moreefficacious than topical administration of medicinal gels containingthese proteins.

[0056] Neurologically active proteins, including erythropoietin (EPO),neurotrophic growth factor (NGF), neurotrophic factor 3 (NT3),neurotrophic factor 4 (NT4), brain-derived neurotrophic factor (BDNF),glial cell line derived neurotrophic factor (GDNF), bone morphogeneticprotein (BMP) may be administered directly into neuronal tissue byintracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices.

[0057] As used for administration of EPO, the phrase “therapeuticallyeffective” is generally from about 1 to 10000 I.U./kg, preferably fromabout 50 to 2000 I.U./kg, more preferably from about 50 to 600 I.U./kg,and most preferably from about 50 to 300 I.U./kg body weight especiallywhen erythropoietin is administered subcutaneously. Advantageously, theformulations of the present invention may be administered to aresponding subject at any desired frequency or time interval betweenadministrations without reduced efficacy. In a preferred dosing regimen,the subject is administered the sustained release formulations of thepresent invention thrice per two weeks, once per week, once per twoweeks, once per three weeks, once per month, once per five weeks, onceper six weeks, or at more frequent or less frequent intervals, or at anycombination of frequencies or time intervals as desired. The effectivedaily dosing of erythropoietin (EPO) is preferably from about 4000 toabout 9000 I.U. (equivalent to about 60,000 I.U. to about 120,000 I.U.every two weeks). Most preferably the effective daily dosing oferythropoietin (EPO) is 5715 I.U. (equivalent to about 80,000 I.U. everytwo weeks). A preferred dosing regimen may be once per three weeks,particularly for subjects receiving chemotherapy for the treatment ofcancer, since many chemotherapeutic regimens are administered on a onceper three-week schedule. However, any dosing schedule of a therapeuticprotein, such as EPO, formulated according to the present invention, canbe easily coordinated with regular visits to the treating physician orwith the dosing schedule of another agent, such as an anti-tumor agent,as is desirable for the patient. This allows the EPO regimen and thechemotherapeutic regimen to be administered simultaneously or inparallel, providing an economic and desirable benefit for the subject.EPO administration is delayed or withheld if the patient, male orfemale, exhibits a hemoglobin level in excess of about 18 g/dL for ahuman male and about 16 g/dL for a human female.

[0058] The following examples are provided for the purpose ofillustrating the present invention, without, however, limiting thepresent invention to the illustrative examples.

EXAMPLE 1 EVALUATION OF EPO SUSTAINED RELEASE FORMULATIONS INIMMUNOSUPPRESSED DOGS

[0059] This study was designed to evaluate CMC-EPO in beagle dogs duringvarious dosing regimens. Pharmacodynamics and pharmacokinetic profilesof EPO (erythropoietin) formulations were also examined.

[0060] Formulations

[0061] EPREX: (Control formulation)

[0062] CMC 1: 15,000 IU EPO, 0.5% CMC, 10 mM NaPhosphate buffer, pH 7,15mg/ml Glycine; Total tonicity=199 mg Pro/% CMC=250

[0063] CMC 2: 15,000 IU EPO, 1.0% CMC, 10 mM NaPhosphate buffer, pH 7,15mg/ml Glycine; Total tonicity=199 mg Pro/% CMC=125

[0064] CMC 3: 15,000 IU EPO, 2.0% CMC, 10 mM NaPhosphate buffer, pH 7,15mg/ml Glycine; Total tonicity=199 mg Pro/% CMC=63

[0065] CMC 4: 15,000 IU EPO, 2.0% CMC, 10 mM NaPhosphate buffer, pH 7,50mg/ml Mannitol; Total tonicity=274 mg Pro/% CMC=63

[0066] Storage

[0067] EPO formulations were stored refrigerated (˜4° C.) protected fromlight when not used on study. Study Animals Species: Dog Strain: BeagleSex: Male Source: Harlan Sprague Dawley, Inc. Indianapolis, Indiana46299 Age at Dosing: 9 to 18 months Target Weight at First Dosing: 6 to18 kg Identification Method: Tattoo applied by animal supplier. Numberon Study: 21 (N = 3 dogs/group)

[0068] Housing

[0069] Dogs were group housed in a dog holding room and acclimated tohandling and sample collection prior to dose administration. Quarantinewas held at least five days prior to dose administration. At the end ofthe quarantine period, the health of all animals was confirmed by studypersonnel. During the study/collection period, the dogs were grouphoused unless necessary due to health conditions. Housing rooms werelabeled with the animals' and protocol numbers.

[0070] Environmental Conditions

[0071] Animal rooms were maintained at 23±3° C. with a relative humidityof 50±15% and a twelve-hour light/dark cycle. There were at least tenroom air changes per hour.

[0072] Diet and Water

[0073] Dogs had access to Purina Certified Canine Diet® #5007 and waterad libitum when on study.

[0074] Study Design (Summary)

[0075] Beagle dogs (N=3 dogs per group, 7 groups) were randomly assignedto treatment groups. On Day −2, the dogs were administered a single oraldose of cyclosporin (25 mg/kg). Thereafter, the dogs received a dailymaintenance dose of cyclosporin (10 mg/kg). All formulations and vehiclewere administered subcutaneously (sc).

[0076] Preparation and Test Formulations Dose Administration: (Day 1)

[0077] Test and control formulations were administered to the dogs onDay 1 (all formulations administered at the volume specified in thefollowing table). The dose was drawn-up into a syringe fitted withappropriate gauge needle. The subcutaneous dose was administered in thedorsal neck region. Dose sites were clipped prior to dosing and markedwith indelible ink. EPO Dose Volume to (IU/kg) Formulations Amount to beInjected be Injected 1,500 Eprex 15,000 IU—every 2 weeks 1 mL/animalDays 1 and 15 1,500 CMC 1 15,000 IU—every 2 weeks 1 mL/animal Days 1 and15 1,500 CMC 2 15,000 IU—every 2 weeks 1 mL/animal Days 1 and 15 1,500CMC 3 15,000 IU—every 2 weeks 1 mL/animal Days 1 and 15 1,500 CMC 415,000 IU—every 2 weeks 1 mL/animal Days 1 and 15

[0078] Observations Sample Collection, and Processing

[0079] At designated primary time points (see below), approximately 2 mLof blood was collected via the jugular vein into heparinizedVacutainers®. In case of jugular vein failure, blood was collected viathe cephalic vein and noted. The primary blood collection, obtainedprior to cyclosporin administration, was used to harvest plasma. Theblood was placed on ice, centrifuged (1500×g, for ten minutes atapproximately 4° C.), and plasma collected. Plasma will be frozen at −20° C. until analysis.

[0080] At the secondary blood collection, approximately 2 mL of bloodcollected using a Vacutainer® containing EDTA was obtained in themorning and placed on ice. The secondary collection was stored atapproximately 4° C. as whole blood and used for reticulocyte,hemoglobin, and total red blood cell measurements.

[0081] Primary Blood Collection Time Points:

[0082] Day 1 (pre-dose, 3, 6, 12, and 16 hours), Day 2 (24 and 36 hours)Day 3, Day 5, Day 7, Day 10, Day 15 (pre-dose), Day 16, and Day 28.

[0083] Secondary Blood Collection Time Points:

[0084] Pre-dose on Days 1, 3, 7, 10, 14, 17, 21, 24, and 28

[0085] Data Analysis

[0086] Excel (Microsoft®, Version 97-SR-2) was used for data processing.WinNonlin (Scientific Consulting®, Version 2.1) was used for processingplasma data and calculating the following pharmacokinetic parameters:C_(max), T_(max), terminal T_(1/2), and Cl/F (Note: the fraction of doseabsorbed cannot be estimated, therefore clearance for this model isactually clearance/F, where F is the fraction of dose absorbed).WinNonlin was also be used to calculate AUC_((0-last)) (of thedifferences from the baseline) for reticulocyte, hemoglobin, and redblood cell data. Applicable statistical analyses (i.e., ANOVA, t-test)were conducted.

[0087] Results

[0088] As seen in FIG. 1, all formulations containing CMC had superiorpharmacokinetic properties compared to EPREX. Notably, the C_(max) waslower than EPREX and the T_(1/2) was longer than that for EPREX.

[0089] As seen in FIGS. 2, 3, and 4, the pharmacodynamic properties ofall CMC formulations, except the formulation containing 50mg/mlmannitol, were superior to EPREX. The formulation containing 50mg/mlmannitol was similar to EPREX. FIG. 2 demonstrates a higher percentchange in reticulocytes following administration of the CMCformulations. Also seen in FIG. 2 is a sharp increase and subsequentdecrease for EPREX following administration of the drug. The decrease ismitigated in all of the CMC formulations, demonstrative of the amount ofsystemic EPO released from the injection site of these formulations.FIGS. 3 and 4 demonstrate the change in Hemoglobin and red blood cell(RBC) versus time. Except for the CMC formulations containing 50mg/mlmannitol and 1% CMC, all other CMC formulations provided superiorpharmacodynamic effects of stimulating hematopoiesis in the dogs.

EXAMPLE 2 EVALUATION OF EPO FORMUALTIONS IN IMMUNOSUPPRESSED DOGS FORONE-MONTH FOLLOWING SUBCUTANEOUS ADMINISTRATION

[0090] Study Design (Summary)

[0091] This study was conducted analogously to that described in Example1, modified as described herein. Beagle dogs (N=3 dogs/group, 5 groups)were randomly assigned to treatment groups. On Day −2, the dogs wereadministered a single oral dose of cyclosporin (25 mg/kg). Thereafter,the dogs received a daily maintenance dose of cyclosporin (10 mg/kg).Two dosing regimens were examined in immunosuppressed dogs. Bothformulations and vehicle were administered in a single subcutaneous (sc)dose. At designated times over a four-week period, blood samples werecollected. The injection site was monitored daily and body weights wereobtained weekly.

[0092] Formulations

[0093] EPREX: 40,000 dose

[0094] CMC: 5 40,000 IU EPO, 7% CMC, 20 mM Na Phosphate buffer, pH 7, 5mg/ml Glycine (0.5%), 75 mM NaCl, 0.3% Tween 80. Total tonicity=142 mMμg Pro/% CMC=47

[0095] Preparation and Test Formulations Dose Administration: (Day 1)

[0096] All formulations were administered at the volume specified in thefollowing table. Dose was drawn-up into a syringe fitted withappropriate gauge needle. The subcutaneous dose was administered in thedorsal neck region. Dose sites were clipped prior to dosing and markedwith indelible ink. Treatments EPO Dose (IU) Dose Volume (mL) Dose RouteEPREX 30,000 0.75 subcutaneous CMC-5 30,000 0.75 subcutaneous

[0097] Primary Blood Collection Time Points:

[0098] All Groups: Pre-dose, 6, 12, and 16 hours on Day 1; 24 and 36hours on Day 2; and Pre-dose on Days 3, 5, 7, 13, 17, 21, 24, and 28.

[0099] Secondary Blood Collection Time Points:

[0100] All Groups: Pre-dose on Days 1, 3, 7, 10, 14, 17, 21, 24, and 28.

[0101] Results

[0102] The CMC-5 formulation demonstrated superior pharmacokineticproperties to EPREX, characterized by a lower C_(max) and a longerT_(1/2). This formulation also demonstrated to have slightly betterpharmacodynamic properties than EPREX.

EXAMPLE 3 EVALUATION OF A CMC-EPO FORMULATION FOLLOWING SUBCUTANEOUSDOSING EVERY THREE WEEKS IN NON-IMMUNOSUPPRESSED MALE BEAGLE DOGS

[0103] Formulation

[0104] CMC-6: 80,000 IU/mL EPO, 1% CMC, 20 mM Na Phosphate pH 7.2, 100mM NaCl; Total tonicity=100 mM μg Pro/% CMC=660

[0105] EPREX: 80K 80,000 IU/mL dose

[0106] Study Overview

[0107] The test and control articles were administered by multiplesubcutaneous injections to beagle dogs, one injection per three-weekperiod. Dose EPO Number of Animals (Male) Dose Volume Strength DosePharmacokinetic Pharmacodynamic Group Treatment (IU/kg) (mL/kg) (IU/mL)Total Fays Sample Days Sample Days 1 CMC-6 1200 0.015 80,000 3 1, 22 1,2, 3, 3.5, 4, 1, 3, 7, 10, 14, 4.5, 5, 7, 10, 17, 21 24, 28, 15, 22, 23,28, 31, 35, 38 and 36 and 42 42 2 Eprex 1200 0.015 40,000 3 1, 22 1, 2,3, 3.5, 4, 1, 3, 7, 10, 14, 4.5, 5, 7, 10, 17, 21, 24, 28, 15, 22, 23,28, 31, 35, 38 and 36 and 42 42

[0108] Pharmacokinetics

[0109] The maximum mean plasma concentrations (C_(max)) and their timesof occurrence (T_(max)) of EPO were determined. The areas under the meanplasma concentration-time curves from time zero to Day 22 (AUC_(0-T)),and their variances, was estimated by the linear trapezoidal ruleaccording to Bailer, J. Pharmacokin. Biopharm., (1988) 16:303-309.Apparent clearance (CL/F) was estimated by dividing AUC_(0-T) by dose(1200 IU/kg). Where appropriate, terminal rate constants (k) wereestimated by fitting a linear regression of log mean concentrationagainst time using data points randomly distributed about a singlestraight line. Terminal half-lives were calculated as In2/k.

[0110] Pharmacodynamics

[0111] Blood for hematology studies (approximately 0.5 mL) was collectedinto tubes containing EDTA anticoagulant as described in the precedingtable.

[0112] The following hematological properties were determined toevaluate the pharmacodynamic properties of the CMC-6 and controlformulations:

[0113] Erythrocyte Count

[0114] Hemoglobin

[0115] Leukocyte Count (total)

[0116] Reticulocyte Count (absolute and percentage)

[0117] Reticulocyte Hemoglobin

[0118] The changes from baseline in the pharmacodynamic parameters (%reticulocytes, hemoglobin, total red blood cells) was calculated usingthe value on Day 1 as the baseline value. The AUC of the change inpharmacodynamic values from baseline was calculated using lineartrapezoidal rule.

[0119] Results

[0120] The pharrnacokinetics of CMC-6 demonstrated a lower C_(max) and alower T_(1/2) than EPREX control. The pharmacodynamics of CMC-6demonstrated a superior change in reticulocyte production by Day 6, butsimilar pharmacodynamic properties to EPREX in all other measures.

EXAMPLE 4 43-DAY PHARMACOKINETIC/PHARMACODYNAMIC STUDY FOLLOWINGSUBCUTANEOUS ADMINISTRATION OF EPO FORMULATIONS IN NON-IMMUNOSUPPRESSEDMALE BEAGLE DOGS.

[0121] This study was designed to assess the pharmacokinetics andpharmacodynamics of the test articles in the male beagle dogadministered via subcutaneous injection. Test System Species/breed:beagle dogs Supplier: Marshall Farms, 5800 Lake Bluff Road North Rose,New York 14516 Number of animals in the study: 18 males Age(approximate) at initiation 5 to 7 months of treatment: Body weightrange (approximate) 8 to 11 kg at initiation of treatment:

[0122] This study was conducted in dogs to evaluate differentformulations of EPO (Epoetin alfa), designed to maintaintreatment-related erythropoiesis, which are for potential registrationwith regulatory agencies for therapeutic use in humans. The number ofdogs used was the minimum number necessary to provide scientificallyvalid results for each of the formulations examined. No acceptable invitro models are available. Purpose-bred beagle dogs are routinely usedfor the conduct of pharmacokinetic, pharmacodynamic, and toxicologicalstudies to meet regulatory requirements.

[0123] Animal Husbandry

[0124] Housing: one room for the study, in an air-conditioned building:

[0125] temperature: 19 to 25° C. (target range)

[0126] relative humidity: >40 %,

[0127] air changes: minimum ten air changes per hour,

[0128] lighting cycle: twelve hours light (artificial)/twelve hoursdark.

[0129] Caging: animals housed singly in pens (1.44 m²).

[0130] Diet: pelleted complete commercial diet (Diet 125C1, UAR);analyzed for the absence of chemical and bacteriological contaminants.

[0131] Quantity distributed: 300 g/animal/day (food offered after dosingon treatment days and at approximately the same time of day on otherstudy days).

[0132] Water: mains drinking water, ad libitum, analyzed at least once ayear for chemical contaminants and at least twice a year for bacterialcontaminants (Laboratoire Santé Environnement Hygiène de Lyon).

[0133] Contaminants: no contaminants known to be present in the diet orwater at levels which might interfere with achieving the objective ofthe study. Certificates of analysis for the diet and for the water willbe maintained in the archives of the testing facility.

[0134] Pre-Treatment Procedures

[0135] Animal health procedure:

[0136] Standard canine vaccination and anti-parasite treatment performedby the supplier

[0137] Clinical examination for ill-health on arrival;

[0138] Full clinical examination during the acclimatization period.

[0139] Acclimatization period:

[0140] Two week minimum between animal arrival and start of treatment.

[0141] Identification of the animals:

[0142] Using tattoo on the pinna and microchip implants: ElectronicLaboratory Animal System, ELAMS (Bio Medic Data Systems). Group NumberColor Code Animal Numbers 1 White 271 to 272 2 Green 274 to 276 3 Blue277 to 279 4 Red 280 to 282 5 Yellow 283 to 285 6 Salmon 286 to 288

[0143] Expermental Design

[0144] Allocation to treatment group: performed during theacclimatization period: random allocation prodecure based on body weightclasses.

[0145] Animals were assigned to the following groups: Epoetin alfa Dose(IU/kg/administration) Number of Group Day 1 Day 15 Day 29 Males 1 15001500 1500 3 2 1500 1500 1500 3 3 1500 1500 — 3 4 1500 1500 — 3 5 15001500 — 3 6 1500 1500 — 3

[0146] Administration of the Test Articles

[0147] Route: subcutaneous

[0148] Method:

[0149] bolus injection using a sterile syringe and needle introducedsubcutaneously after local disinfection with an aqueous solution ofethyl alcohol. One injection site in the dorsal cervical/interscapularregions was used.

[0150] Volume administered:

[0151] Individual dose volumes were calculated on the day of treatmentto two decimal places according to the latest recorded body weight. Thesyringes containing the dosing solution were weighed before and afteradministration in order to calculate the actual individual delivereddose.

[0152] Frequency: Once on the day of treatment (Day 1).

[0153] Observations

[0154] Morbidity/Mortality:

[0155] Animals were observed at least twice daily. Any animal judged tobe in a moribund condition will be bled for haematological, clinicalchemistry and pharmacokinetic determinations and then euthanized.

[0156] Clinical Signs

[0157] Animals were observed daily. During the treatment period, animalswere examined before and at least once after dosing to detect anyclinical signs or reaction to treatment. The injections sites wereobserved daily. A full clinical examination were performed before theinitiation of treatment.

[0158] Body Weight: Individual weighing once weekly, starting two weeksbefore initiation of treatment.

[0159] Clinical Laboratory Determinations

[0160] Blood Collection:

[0161] Blood was withdrawn from the jugular vein of the unanaesthetized,manually restrained animal.

[0162] Collection of samples

[0163] Haematology parameters: EDTA.

[0164] Clinical Chemistry parameters: without anticoagulant.

[0165] Haematology

[0166] All animals: twice prior to dosing (Day - 7 and prior to dosingon Day 1), and on Days 3, 5, 7, 9, 11, 13, 15, 18, 21, 24, 27, 30.Animals from Groups 1 and 2 will additionally be bled on days 33, 36, 39and 43.

[0167] Approximately 2 ml of whole blood will be taken from each dog pertime point.

[0168] Hemoglobin

[0169] Red Blood Cell Count

[0170] Mean Corpuscular Volume

[0171] Reticulocyte Count (absolute and relative)

[0172] Total White Blood Cell Count

[0173] Reticulocyte Haemoglobin

[0174] Reticulocyte Sub-Populations (H-, M- and L-reticulocytes)

[0175] Clinical Chemistry

[0176] All animals: prior to dosing (Day 1) and at study termination(Day 30: Groups 3 to 6; Day 43: Groups 1 and 2).

[0177] Approximately 2 ml of whole blood will be taken from each dog pertime point.

[0178] Sodium

[0179] Potassium

[0180] Chloride

[0181] Calcium

[0182] Inorganic phosphorus

[0183] Glucose

[0184] Urea

[0185] Total cholesterol

[0186] Triglycerides

[0187] Total bilirubin

[0188] Total protein

[0189] Albumin

[0190] Globulin (calculated)

[0191] Albumin/globulin ratio

[0192] Creatinine

[0193] Alkaline phosphatase

[0194] Aspartate aminotransferase

[0195] Alanine aminotransferase

[0196] Gamma glutamyl transferase.

[0197] Pharmacokinetics

[0198] Animals examined: all animals.

[0199] Blood was withdrawn from the jugular vein of the unanaesthetized,manually restrained animal. Approximately 2 ml of whole blood were takenfrom each dog per time-point into containers with heparin anticoagulantplaced on wet ice. Plasma samples were obtained by centrifugation atapproximately 3000 rpm for ten minutes and transferred to polypropylenetubes. The plasma samples were stored deep-frozen (approximately −20°C.) pending dispatch to the auxiliary Testing Facility 1.

[0200] Samples were taken at the following time points: Groups 1 and 2Day 1 Pre-dose, 6 and 24 hours after dosing Day 2 24 and 36 hours afterdosing on Day 1 Days 3, 3, 4, Once daily 5, 6, 7 and 8 Day 15 Seconddose (pre-dose) Day 16 Once daily Day 29 Third dose (pre-dose) Day 30Once daily and 43 Groups 3 to 6 Day 1 Pre-dose, 6 and 24 hours afterdosing Day 2 24 and 36 hours after dosing on Day 1 Days 3, 4, 5, Oncedaily 6, 7 and 8 Day 15 Second dose (pre-dose) Day 16 Once daily and 30

[0201] Pharmacodynamic Data Evaluation

[0202] The group mean values for all pharmacodynamic (haematology)parameters were plotted (FIGS. 5 to 8).

EXAMPLE 5 PHARMACOKINETIC/PHARMACODYNAMIC CROSS-OVER G-CSF FORMULATIONSTUDY BY SUBCUTANEOUS ADMINISTRATION IN NON-IMMUNOSUPPRESSED MALE BEAGLEDOGS

[0203] Test System Species/breed: Beagle dogs Supplier: Harlan France,Z. I. du Malcourlet, R.N. 9, B.P. 98, 03800 GANNAT, France Number ofanimals in study: 6 dogs Age (approximate) at initiation 5 to 7 monthsof study: Body weight range (approximately) 9 to 11 kg at initiation ofstudy:

[0204] This study is conducted in dogs to evaluate various formulationsof GCSF (Granulocyte colony stimulating factor). The number of dogs usedwas the minimum number necessary to provide scientifically valid resultsfor each of the formulations examined. No acceptable in vitro models areavailable. Purpose-bred beagle dogs are routinely used for the conductof pharmacokinetic, pharmacodynamic and toxicological studies to meetregulatory requirements.

[0205] Animal Husbandry

[0206] Housing: one room for the study, in an air-conditioned building

[0207] Temperature: 19 to 25° Celsius (target range)

[0208] Relative humidity:>40 %

[0209] Air changes: Minimum 10 air changes per hour

[0210] Lighting cycle: 12 hours light (artificially)/12 hours dark.

[0211] Caging: animals housed singly in pens (1.44 m²)

[0212] Diet: pelleted complete commercial diet (Diet 125C1, UAR),analyzed for the absence of chemical and bacteriological contaminants.

[0213] Quantity distributed: 300 g/animal/day (food is offered afterdosing on treatment days and at approximately the same time of day onother study days).

[0214] Animals are fasted overnight before sampling for clinicallaboratory determinations and before necropsy. Due to the nature of thesampling schedule, not all of the pharmacokinetic samples are takenunder fasted conditions.

[0215] Water: mains drinking water, ad libitum, analyzed at least once ayear for chemical contaminants and at least twice a year for bacterialcontaminants.

[0216] Contaminants: no contaminants are known to be present in the dietor water at levels which might interfere with achieving the objective ofthe study. Certificates of analysis for the diet and for the water ismaintained in the archives of the testing facility.

[0217] Pre-Treatment Procedures

[0218] Animal health procedure:

[0219] Standard canine vaccination and anti-parasite treatment performedby the supplier;

[0220] Clinical examination for ill health on arrival;

[0221] Full clinical examination during the acclimatization period.

[0222] Acclimatization period:

[0223] Two weeks minimum between animal arrival and start of treatment.

[0224] Identification of the animals:

[0225] Using tattoo on the pinna and microchip implants: ElectronicLaboratory Animal Monitoring System ELAMS (Bio Medic Data Systems)Identification numbers. Group Number Color Code Animal Numbers 1 Green391 to 393 2 Blue 394 to 396

[0226] Experimental Design

[0227] Allocation to treatment group: performed during theacclimatization period: random allocation procedure based on body weightclasses.

[0228] Animals are assigned to the following groups: GCSF Dose Treatment(μg/kg/ Number Group Day 1 Day 15 administration)* of males 1 TestArticle A Test Article B 70 3 2 Test Article B Test Article A 70 3

[0229] Article A: GCSF, Lot No. 14901-185, Concentration: 1.164 mg/mlformulated as described herein.

[0230] Article B: CMC-GCSF, Lot No. 15849-136-1, Concentration: 0.60mg/ml formulated as described herein.

[0231] The dose volumes required to perform the above regimen are asfollows: GCSF Dose (μg/kg/administration) Number of Group Day 1 Day 14males 1 0.60 To be defined by 3 protocol amend- ment 2 0.117 To bedefined by 3 protocol amend- ment

[0232] Rationale for Dose Selection: The doses were selected based onexisting data obtained from formulations evaluated in previous studies.

[0233] Administration Of Test Articles

[0234] Method: bolus injection using a sterile syringe and needleintroduced subcutaneously after local disinfection with an aqueoussolution of ethyl alcohol. Two injection sites in the dorsalcervical/interscapular regions is in rotation.

[0235] Volume administered: dependent on dosing regimen. Individual dosevolumes are calculated on Days 1 and 15 to two decimal places accordingto the latest recorded body weight; these volumes are recorded in thestudy data. The syringes containing the dosing solution are weighedbefore and after administration in order to calculate the actualindividual delivered dose.

[0236] Rationale for choice of route of administration: The subcutaneousroute was selected as this is the route of administration in humans

[0237] Route: Subcutaneous

[0238] Frequency: Once on Days 1 and 15.

[0239] Duration: Two administrations over the course of the study (Days1 and 15)

[0240] Morbidity/Mortality

[0241] Animals are observed at lease twice daily. Any animal judged tobe in a moribund condition is bled for haematological, clinicalchemistry and pharmacokinetic determinations and then euthanized.

[0242] Clinical Signs

[0243] Animals are observed daily. During the treatment period, animalsare examined before and at least once after dosing to detect anyclinical signs or reaction to treatment. The injection sites areobserved daily. A full clinical examination is performed before theinitiation of treatment.

[0244] Body Weight

[0245] Individuals weighing once weekly, starting two weeks beforeinitiation of treatment.

[0246] Blood Collection

[0247] Blood:

[0248] Withdrawn from the jugular vein of on unanaesthetized, manuallyrestrained animal.

[0249] Collection of samples:

[0250] Haematology parameters: EDTA

[0251] Clinical Chemistry parameters: Without anticoagulant

[0252] Haematology

[0253] All animals: Twice prior to dosing (Day −7 and prior to dosing onDay 1), and at the following time-points: Day 1 6, 12 and 16 hours aftertreatment 1 Day 2 24 and 36 hours after treatment 1 Day 3 48 hours aftertreatment 1 Day 4 72 hours after treatment 1 Day 5 96 and 108 hoursafter treatment 1 Day 15 6, 12 and 16 hours after treatment 2* Day 16 24and 36 hours after treatment 2* Day 17 48 hours after treatment 2* Day18 72 hours after treatment 2* Day 19 96 and 108 hours after treatment2*

[0254] Approximately 0.5 ml of whole blood is taken from each dog pertime-point.

[0255] Total white blood cell count

[0256] Differential white blood cell count (neutrophils, lymphocytes,monocytes, eosinophils and basophils).

[0257] Quality Control Of Haematological Data

[0258] The quality of haematological data is controlled as follows:

[0259] Controls are run before and after each initial run of bloodsamples (repeat analysis not included). For each control batch, a meanand standard deviation are calculated per time-point for the entirein-house life span of each given batch used during the course of thestudy. The batch numbers are also recorded. The ADVIA, used to performhaematological determinations is operated in CBC/DIFF mode in order tocollect differential white blood cell data.

[0260] Clinical Chemistry

[0261] Performed on all animals prior to dosing on Day 1 and attermination (Day 19). Approximately 2 ml of whole blood is taken fromeach dog per time-point.

[0262] Sodium

[0263] Potassium

[0264] Chloride

[0265] Calcium

[0266] Inorganic phosphorus

[0267] Glucose

[0268] Urea

[0269] Total cholesterol

[0270] Total nilirubin

[0271] Total protein

[0272] Albumin

[0273] Globulin (calculated)

[0274] Albumin/globulin ratio

[0275] Creatinine

[0276] Triglycerides

[0277] Alkaline phosphatase

[0278] Aspartate aminotransferase

[0279] Alanine aminotransferase

[0280] Gamma glutamyl transferase

[0281] Pharmacokinetics

[0282] Animals examined: all animals

[0283] Blood: withdrawn from the jugular vein of the unanaesthatized,manually restrained animal. Approximately 2 ml of whole blood is takenfrom each dog per time-point into containers with heparin anticoagulantplaced on wet ice. Plasma samples are obtained by centrifugation atapproximately 3000 rpm for ten minutes and transferred to polypropylenetubes. The plasma samples are stored in deep-frozen (approximately −20degrees)

[0284] Samples are collected from all animals at the followingtime-points Day 1 Pre-dose, 2, 4, 6, 8, 10, 12 and 16 hours aftertreatment 1 Day 2 24 and 36 hours after treatment 1 Day 3 48 hours aftertreatment 1 Day 4 72 hours treatment 1 Day 5 96 and 108 hours aftertreatment 1 Day 15 Pre-dose, 2, 4, 6, 8, 10, 12 and 16 hours aftertreatment 2* Day 16 24 and 36 hours after treatment 2* Day 17 48 hoursafter treatment 2* Day 18 72 hours after treatment 2* Day 19 96 and 108hours after treatment 2*

[0285] Data Evaluation

[0286] Pharmacodynamic Data Evaluation:

[0287] The group mean values for all phamacodynbamic (haematology)parameters is plotted as graphs. The areas under the parameter-timecurves (AUC_((0-last)) of the baseline adjusted parameter) are estimatedby the linear trapezoidal rule according to Bailer (1988). Reference forthis technique is A. J. Bailer, J. Pharmacokin. Biopharm., (1988)1:303-309. Only positive areas under the mean parameter-time curves arecalculated using the following rules:

[0288] 1) each individual parameter baseline is determined on acase-by-case basis using the value from either Day 1 or Day 3, the lowervalue of the two setting the baseline.

[0289] 2) Where the curve falls below the baseline, the time-point(s)concerned are assigned the baseline value; this only applies to(AUC_((0-last)) calculation.

[0290] Arithmetic group mean and standard deviation values arecalculated for each parameter and its respective (AUC_((0-last))

[0291] Pharmacokinetic Data Evaluation

[0292] The maximum mean plasma concentrations (C_((max))) and theirtimes of occurrence (T_((max))) are the observed values. The areas underplasma concentration-time curves (AUC_(t)), and their variances, areestimated by the linear trapezoidal rule according to Bailer (1988).Where appropriate, terminal rate constants (k) are estimated by fittinga linear regression of log mean concentration against time using datapoints randomly distributed about a single straight line. Terminalhalf-lives are calculated as Ln2/k together with clearance (Cl+achieveddose/AUC_(t)). Reference for this technique is A. J. Bailer, J.Pharmacokin. Biopharm., (1988) 16:303-309.

What is claimed is:
 1. A pharmaceutical formulation comprising: a) a pharmaceutically active amount of erythropoietin; b) a pharmaceutically acceptable pH buffering agent to provide a pH in a range of about pH 6 to about pH 9; c) a tonicity agent in a concentration range of about 0 to about 200 millimolar; and d) sodium carboxymethyl ether cellulose in a concentration range of about 0.5% to about 7% total formula weight, said CMC having a molecular weight in a range of about 50,000 daltons to about 1,000,000 daltons.
 2. The formulation of claim 1 wherein the erythropoietin is selected from a group consisting of recombinant human erythropoietin, epoietin alfa, epoietin omega, darbepoetin alfa, and PEG conjugated erythropoietin.
 3. The formulation of claim 1 wherein the pH buffering agent concentration is in the range of about 10 mM to about 30 mM and wherein the pH buffering agent is a sodium phosphate monobasic/sodium phosphate dibasic buffer system.
 4. The formulation of claim 1 wherein the tonicity agent is selected from a group consisting of NaCl, KCl, and glycine.
 5. The formulation of claim 1 wherein the tonicity agent is NaCl and the NaCl concentration is about 75 mM to about 125 mM.
 6. The formulation of claim 1 wherein the pH of the formulation is in the range of about 6.5 to about 7.4.
 7. A method of treating a subject in need of such treatment comprising administering to the subject a pharmaceutical formulation according to claim
 1. 8. The method of claim 7 wherein the pharmaceutical formulation is administered in accordance with a regimen of administering thrice per two weeks, once per week, once per two weeks, once per three weeks, once per month, once per five weeks, or once per six weeks.
 9. The method of claim 8 wherein the effective daily dosing of erythropoietin is from about 4000 to about 9000 I.U.
 10. The method of claim 9 wherein the effective daily dosing of erythropoietin is greater than 10,000 I.U.
 11. A method of formulation comprising the steps, in any order: a) providing a pH buffered erythropoietin sample; b) admixing a quantity of CMC to the pH buffered erythropoietin sample sufficient to provide a final concentration of about 0.5% to about 7% CMC total formula weight; c) admixing a quantity of NaCl to the pH buffered erythropoietin sample sufficient to provide a final concentration of about 0 mM to about 170 mM NaCl; and d) adjusting the pH buffered erythropoietin sample with water sufficient to provide a predetermined final formulation volume and erythropoietin potency.
 12. A pharmaceutical formulation comprising: a) a pharmaceutically active amount of a protein; b) a pharmaceutically acceptable pH buffering agent to provide a pH in the range of about pH 4.5 to about pH 9; c) a tonicity agent in the concentration range of about 0 to about 200 millimolar; and d) Sodium carboxymethyl ether cellulose in the concentration range of about 0.5% to about 7% total formula weight, said CMC having a molecular weight in the range of about 50,000 daltons to about 1,000,000 daltons.
 13. The formulation of claim 12 wherein the protein is selected from a group consisting of insulin, motilin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), growth hormone (GH), kerantinocyte growth factor (KGF), stem cell factor (SCF), thrombopoietin, osteoprotegerin (OPG), obesity protein (OB protein), leptin, granulocyte colony-stimulating factor (G-CSF), alpha interferon, beta interferon, gamma interferon, interleukin 2, fibroblast growth factors (FGF), insulin-like growth factors (IGF), macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), colony simulating growth factors (CSFs), tumor necrosis factor (TNF), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), neurotrophic growth factor (NGF), neurotrophic factor 3 (NT3), neurotrophic factor 4 (NT4), brain-derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF), platelet-derived growth factor (PGDF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), megakaryocyte growth differentiation factor (MGDF), Factor VII, Factor VIIa, Factor VIII, Factor IX, superoxide dismutase (SOD), tissue plasminogen activator (TPA), urokinase, streptokinase, kallikrein, alpha-galactosidase, pancreatic RNAase, platelet activating factor acetylhydrolase, interleukin-1 receptor antagonist (IL-Ira), Infliximab, antibodies, and etanercept.
 14. The formulation of claim 12 wherein the pH buffering agent concentration is in the range of about 10 mM to about 30 mM and wherein the pH buffering agent is a sodium phosphate monobasic/sodium phosphate dibasic buffer system.
 15. The formulation of claim 12 wherein the tonicity agent is selected from a group consisting of NaCl, KCl, and glycine.
 16. The formulation of claim 12 wherein the tonicity agent is NaCl and the NaCl concentration is about 75 mM to about 125 mM.
 17. The formulation of claim 12 wherein the pH of the formulation is in the range of about 6.5 to about 7.4.
 18. A method of treating a subject comprising administering to the subject a pharmaceutical formulation according to claim
 12. 19. The method of claim 18 wherein the pharmaceutical formulation is administered in accordance with a regimen of administering thrice per two weeks, once per week, once per two weeks, once per three weeks, once per month, once per five weeks, or once per six weeks. 